Programmable one-pot polymerase-mediated DNA synthesis via temperature control

Polymerase-mediated DNA synthesis is fundamental to numerous biotechnology applications, but existing programmable synthesis methods depend on exchanging DNA building blocks, thereby increasing reagent use and complicating multistep workflows. Here, we introduce the TEmperature Mediated Primer Exchange Reaction (TEMPER), a programmable platform for arbitrary DNA synthesis that operates solely through temperature control without solution exchange. TEMPER uses hairpin DNA as temperature-responsive building blocks that define specific temperature range for DNA synthesis. The temperature range is determined by the length design of the hairpin, which regulates thermodynamic interactions between DNA molecules and allows selective and sequential DNA synthesis in one-pot. We validate its versatility by developing a DNA data storage writer, a colorimetric temperature indicator, and a temperature data logger, highlighting its broad potential in nanotechnology and biotechnology applications.